1. Field of the Invention
The present invention relates to novel radiolabled compounds, and to a process for their manufacture. The invention is further directed to compositions comprising these labeled compounds, as well as to methods of using these compounds.
2. Related Art
xcex3-Aminobutyric acid (GABA) receptors are intrinsic membrane glycoproteins in vertebrate and invertebrate neuronal tissues that are members of the ligand-gated ion channel superfamily of receptors. GABA receptors play a major role in the inhibition of central nervous system (CNS) neuronal activity due to the widespread distribution of GABA-releasing and GABA-receptive neurons.
Vertebrate GABA receptors can be divided into two major classes: the GABAA and GABAC receptor subtypes, and GABAB receptor types, which are distinguished by differences in their effector mechanisms and pharmacology (Knapp, R. J., et al., Neurochem. Res. 15:105-112 (1990)). GABAA and GABAC receptors are transmitter-operated chloride channels that are activated by GABA to open their chloride channel while GABAB receptors are thought to mediate changes in cyclic AMP levels through the activation of phospholipase activity (Eldefrawi, A. T. and Eldefrawi, M. E., FASEB J. 1:262-271 (1987); Knapp, R. J., et al., Neurochem. Res. 15:105-112 (1990)). The GABAA receptor and its associated chloride ion channel make up the so-called GABAA receptor-channel complex.
GABA receptors play an important role in the chemical control of insects, such as fleas, ticks, house flies, fruit flies, plant bugs, boll weevils, grasshoppers, cockroaches, mosquitoes, beetles, locust and moths (Hainzl, D., et al., Chem. Res. Toxicol. 11: 1529-1535 (1998)). To date, all insect GABA receptors studied gate a fast acting chloride ion conductance. Although they appear to share many of the properties of GABAA-type receptors in the vertebrate CNS, the majority of receptors in the insect nervous system appear to be bicuculline-, pitrazepin- and RU5135-insensitive (Anthony, N. M., et al., Comp. Mol. Neurobiol., Pichon, Y., ed., Birkhxc3xa4user Verlag, Basel, Switzerland, pp. 172-209 (1993); Wafford, K. A., et al., J. Neurochem. 48:177-180 (1987)). These findings indicate that insect GABA receptors contain several drug binding sites with structural and target site specificities that are different from vertebrate receptor-binding sites (Hainzl, D., et al., Chem. Res. Toxicol. 11:1529-1535 (1998)). Selective insecticides, e.g., insecticides with favorable selective toxicity for insects relative to vertebrates, are based in part on this target-site specificity between the GABA receptors of insects and GABAA receptors of vertebrates (Moffat, A. S., Science 261:550-551 (1993); Hainzl, D., et al., Chem. Res. Toxicol. 11:1529-1535 (1998)).
Radiolabeled ligand binding studies have expanded our knowledge of insect GABA receptor pharmacology. Within the GABA receptor three distinct binding sites have been identified: the GABA receptor agonist binding site, a benzodiazepine binding site and a convulsant binding site (Lummis, S. C. R., Comp. Biochem. Physiol. 95C:1-8 (1990); Rauh, J. J., et al., TiPS 11:325-329 (1990)). The convulsant binding site of GABA receptors in insects is the major target site for many of the drugs and insecticides currently on the market.
Convulsant drugs and insecticides act at the GABA receptor in insect brain, ganglia and muscle as noncompetitive blockers. Inhibition of GABA receptors in insects produces neurotoxicity (e.g., convulsions, paralysis, coma and death). In the early 1980s, the insecticides lindane and cyclodienes (e.g., dieldrin) were shown to antagonize the action of GABA in stimulating chloride uptake by various insect nerve and muscle preparations (Narahashi, T., Pharmacol. Toxicol. 78:1-14 (1996)). GABA receptors in insects are also blocked by picrotoxin, phenylpyrazole insecticides (e.g., Fipronil(copyright)), bicyclophosphorous esters (e.g., t-butylbicyclophosphoronthionate), and bicycloorthobenzoates (4-n-propyl-4xe2x80x2-ethynylbicycloorthobenzoate) (U.S. Pat. No. 5,853,002). These insecticides block transmission of signals by GABA, and are very effective on a wide range of economically important pests.
Unfortunately, many potent insecticides and their derivatives also act at the GABAA receptors of host animals. For example, fipronil sulfone and desulfinyl fipronil, a metabolite and photoproduct of fipronil, respectively, are not only toxic to insects, but also to upland game birds, freshwater fish and invertebrates, and waterfowl. In addition, fipronil itself is a toxicant for mammals even without oxidation to the sulfone (Hainzl, D., et al., Chem. Res. Toxicol. 11:1529-1535 (1998)).
Target-site specificity between the GABA receptors of insects and mammals can be assayed with the radioligand 4xe2x80x2-ethynyl-4-n-[2,3-3H2]-propylbicycloorthobenzoate ([3H]EBOB, 2). Compounds 3-5, were also developed as radioligands for the GABA-gated chloride channel. Casida,, J. E., Arch. Insect Biochem. and Physiol. 22:13 (1993); and Palmer, C. J. and Casida, J. E., J. Labelled Compounds and Radiopharmaceuticals 29:829 (1991).
The development of suitable radioligands constitutes a major step in deepening the understanding of the GABA-gated chloride channel. 
Radioligands 3-5, among others, have proven unsatisfactory for the insect receptor. This was attributed to their low insecticidal activity. Radioligand 2 is presently used. However, it is unstable and must be periodically purified.
Insect neuronal GABA receptors exhibit pharmacological similarity to mammalian GABAA receptors, but also exhibit critical differences, for example in the potency order for agonists and antagonists. Adverse toxicology has led to significant restrictions on the use of many insecticides. Further, the selection of resistant pest strains has resulted in the ineffectiveness of some chloride channel blockers and activators as insecticides. A better understanding of the insect GABA receptor is necessary to develop insecticides that are not subject to resistance, and that exhibit better selectivity and enhanced environmental safety.
A need exists in the art for a radiolabeled compound that binds to the pest GABA gated chloride ion channel, and possesses chemical stability.
A first aspect of the present invention is directed to radiolabeled compounds of Formula I.
A second aspect of the invention is directed to a method of demonstrating specific pest GABA receptors, qualitatively screening for compounds acting on a pest GABA receptor or quantitatively assaying concentrations of a compound acting on a pest GABA receptor, comprising contacting a pest GABA receptor with a compound of Formula I, or a salt thereof.
A third aspect of the invention is directed to a composition comprising at least one compound of Formula I, or a salt thereof, and an acceptable carrier or diluent.
A fourth aspect of the present invention is directed to methods for synthesizing radiolabeled compounds of Formula I.